Deprenyl compounds for treatment of glaucoma

ABSTRACT

Methods and kits for treament of glaucoma are disclosed. In general, the methods of the invention include administering a therapeutically effective amount of a deprenyl compound to a subject such that the subject is treated for glaucoma.

This application is a continuation application of U.S. Ser. No.09/119,337 filed on Jul. 20, 1998, now U.S. Pat. No. 5,981,598 which isa continuation of U.S. Ser. No. 08/598,845 filed Feb. 9, 1996 now U.S.Pat. No. 5,783,606, which is a continuation-in-part of 08/515,893, filedAug. 16, 1995 (abandoned), which is a continuation of 08/394,003, filedFeb. 10, 1995 (abandoned). This application also claims priority to U.S.Ser. No. 09/097,367, filed Jun. 15, 1998 and U.S. Ser. No. 08/599,009,now U.S. Pat. No.5,844,003, which is a continuation-in-part of U.S. Ser.No. 08/470,301, filed Jun. 6, 1995 (abandoned), which is a continuationof U.S. Ser. No. 08/374,332, filed Jan. 18, 1995, now U.S. Pat.No.5,767,164, which is a continuation of 08/203,726, filed Feb. 28,1994, now U.S. Pat. No.5,444,095, which is a continuation of U.S. Ser.No. 07/929,579, filed Aug. 14, 1992 (abandoned), which is acontinuation-in-part of U.S. Ser. No. 07/772,919, filed Oct. 8, 1991(abandoned), which is a continuation-in-part of U.S. Ser. No.07/751,186, filed Aug. 26, 1991 (abandoned), which is acontinuation-in-part of U.S. Ser. No. 07/678,873, filed Apr. 4, 1991(abandoned). The contents of all of the aforementioned applications andissued patents are hereby expressly incorporated by reference.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 08/515,893,entitled “A Method of Treating Optical Nerve Damage with Deprenyl”,filed on Aug. 16, 1995, which is a continuation of U.S. Ser. No.08/394,003, entitled “A Method of Treating Optical Nerve Damage withDeprenyl”, filed on Feb. 10, 1995, now abandoned. The methods of thisapplication also can involve compounds and/or compositions described inU.S. Pat. No. 5,449,095, and a co-pending application entitled “Use ofDeprenyl Compounds to Maintain, Prevent Loss, or Recover Nerve CellFunction”, filed on even date herewith. The contents of all of theaforementioned applications and issued patents are hereby expresslyincorporated by reference.

BACKGROUND OF THE INVENTION

Glaucoma is a disease of the eye characterized by elevated intraocularpressure. The elevated intraocular pressure leads to hardening of theeyeball, narrowing of the field of vision and a decrease in a subject'svisual acuity. Glaucoma is a disease of the optic nerve and the elevatedeye pressures are related to damage of this nerve. The optic nervecarries images from the retina to the brain. Glaucoma damages opticalnerve cells causing blindspots to occur within a subject's vision. Theseblind spots typically are not noticed by the subject until considerabledamage to the optic nerve has already occurred. The terminal stage ofglaucoma is total blindness of the subject.

Approaches to treating glaucoma include the topical application ofcholinergic agents, e.g., pilocarpine, alpha- or beta- adrenergicagonists or antagonists, e.g., clonidine, timolol or epinephrine. Analternative approach for treating glaucoma is the systemicadministration of carbonic anhydrase inhibitors. In some cases laser oroperative surgery is used to treat glaucoma.

Problems exist with the aforementioned approaches to treating glaucomain that the treatments can be accompanied by side-effects. For examplethe instillation of a cholinergic agent, such as pilocarpine, into theeye of a subject can cause nausea, diarrhea, muscular spasms, sweating,lacrimation, salivation, etc. Contraction of the pupil (myosis) and ofthe ciliary muscle of the eye, as well as dilation of the blood vesselsof the iris and conjunctiva also can be observed. Visual complications,e.g., spasm of accommodation, myopia or a decrease in visual acuity,also can occur.

The treatment with a sympathomimetic agent such as dipivalylepinephrineis known frequently to produce sensations of burning or irritation in asubject. Another side-effect of these agents is the appearance ofcardiac disturbances, e.g., palpitations, tachycardia, arrythmia, etc.

Clonidine, which is known as an alpha-2-adrenergic receptor agonist, canbring about mydriasis, as well as an initial phase of ocularhypertension (biphasic effect). Furthermore, in spite of the topicalapplication of the product to the eye, important systemic effects, suchas bradycardia and hypotension, have been observed.

The use of beta-blocking medicaments also can cause important systemiceffects after topical administration to the eye, due to the absence of a“first pass effect”. Timolol, for example, causes bradycardia orhypotension. These systemic secondary reactions to beta-blockingmedicaments can reach such a severe level that the treatment has to bediscontinued. Cases of suicidal depression, hallucinations, nightmaresor psychoses requiring hospitalization have been reported in connectionwith these medicaments. Furthermore, these compounds have to beadministered with extreme precautions to patients subject to cardiac orpulmonary functional disorders. In such patients, amongst others, casesof arrhythmia, cardiac arrest, asthma, dyspnea and bronchospasms havebeen reported.

The treatment with a sympatholytic agent, such as guanethidine, causeshyperemia of the conjunctiva and some irritation, not to mention thefact that these agents only have a low tendency to reduce intraocularpressure.

Finally, in the treatment of glaucoma with carbonic anhydraseinhibitors, such as acetazolamide or methazolamide, serious systemicside-effects, such as depression of the central nervous system, weightloss and, mainly, bone marrow hypofunction, have been reported.

The use of conventional hypotensive agents for the treatment of glaucomais accompanied by considerable risks. Known medications are notparticularly well suited for topical treatment and the systemicside-effects of these medicaments make them delicate to use becausethese effects are far from being negligible and because they can have,in some cases, severe consequences.

SUMMARY OF THE INVENTION

This invention provides methods and kits useful for the treatment ofglaucoma. In one aspect, the methods of the invention includeadministering a therapeutically effective amount of a deprenyl compoundto a subject such that the subject is treated for glaucoma. In apreferred embodiment, the deprenyl compound is represented by thestructure:

in which R₁ is hydrogen, alkyl, alkenyl, alkynyl, aralkyl,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, or aryloxycarbonyl; R₂ ishydrogen or alkyl; R₃ is a single bond, alkylene, or—(CH₂)_(n)—X—(CH₂)_(m) in which X is O, S, or N-methyl; m is 1 or 2, andn is 0,1, or 2; R₄ is alkyl, alkenyl, alkynyl, heterocyclyl, aryl oraralkyl; R₅ is alkylene, alkenylene, alkynylene and alkoxylene; and R₆is C₃-C₆ cycloalkyl or

—C≡CH; or

R₂ and R₄-R₃ are joined to form, together with the methine to which theyare attached, a cyclic or polycyclic group; and pharmaceuticallyacceptable salts thereof. In preferred embodiments, R₁ is a group thatcan be removed in vivo; R₁ is hydrogen; R₁ is alkyl; R₁ is methyl; R₂ ismethyl; R₃ is methylene; R₄ is aryl; or R₄ is phenyl. In still otherpreferred embodiments, R₅ is alkylene, more preferably methylene. Inother preferred embodiments, R₆ is —C≡CH. In other preferredembodiments, R₆ is cyclopentyl.

In another embodiment, the deprenyl compound has the structure

in which R₁ is hydrogen, alkyl, alkenyl, alkynyl, aralkyl,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, or aryloxycarbonyl.

In another preferred embodiment, the deprenyl compound is represented bythe structure:

in which R₁ is hydrogen, alkyl, alkenyl, alkynyl, aralkyl,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, or aryloxycarbonyl; R₂ ishydrogen or alkyl; R₃ is a bond or methylene; and R₄ is aryl or aralkyl;or R₂ and R₄-R₃ are joined to form, together with the methine to whichthey are attached, a cyclic or polycyclic group; and pharmaceuticallyacceptable salts thereof.

In another embodiment, the deprenyl compound is represented by thestructure:

in which

R₂ is hydrogen or alkyl; R₃ is a bond or methylene; and R₄ is aryl oraralkyl; or R₂ and R₄-R₃ are joined to form, together with the methineto which they are attached, a cyclic or polycyclic group; and R₅ isalkylene, alkenylene, alkynylene and alkoxylene; and pharmaceuticallyacceptable salts thereof.

In yet another embodiment, the deprenyl compound is represented by thestructure:

in which R₁ is hydrogen, alkyl, alkenyl, alkynyl, aralkyl,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, or aryloxycarbonyl; A is asubstituent independently selected for each occurence from the groupconsisting of halogen, hydroxyl, alkyl, alkoxyl, cyano, nitro, amino,carboxyl, —CF₃, or azido; n is 0 or an integer from 1 to 5; andpharmaceutically acceptable salts thereof.

In other preferred embodiments, the deprenyl compound is (−)-deprenyl,(−)-pargyline, or (−)-desmethyldeprenyl.

In another aspect, the invention provides a kit useful for the treatmentof glaucoma. In one embodiment, the kit includes a container of adeprenyl compound and instructions for administering a therapeuticallyeffective amount of the deprenyl compound to a subject such that thesubject is treated for glaucoma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a box plot of the distributions of the counts of retinalganglion cell layer neuronal cell bodies (RGCLncbs) for fourexperimental groups, showing that deprenyl treatment increases neuronalsurvival after optic nerve crush.

FIG. 2 shows a plot of counts of Nissl stained cell bodies and FGlabelled cell bodies for varying lengths of retinal sections, showingthat neuronal cell bodies in the retinal ganglion cell layer (RGCL) sendaxons to the SC.

FIG. 3 depicts the distributions of surviving retinal ganglion cellsprojecting to the superior colliculus (RGC^(SC)s) as a percentage of thedistribution for uncrushed saline-treated retinas, and showing thatdeprenyl treatment increases survival of RGC^(SC)s after optic nervecrush.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of treating glaucoma. In general,the methods include administering a therapeutically effective amount ofa deprenyl compound to a subject in need thereof, such that the subjectis treated for glaucoma.

The language “glaucoma” is art-recognized. The term includes both acuteand chronic diseases of the eye characterized by elevated intraocularpressure. Symptoms of glaucoma include high pressure within the eyeball,hardening of the eyeball, narrowing of the field of vision, death ofoptic nerve cells, development of retinal blind spots, and decreasedvisual acuity.

The term “subject”, as used herein, refers to a warm-blooded animal inneed of treatment for, or susceptible to, glaucoma. In preferredembodiments, the subject is a mammal, including humans and non-humanmammals such as dogs, cats, pigs, cows, sheep, goats, rats, and mice. Ina particularly preferred embodiment, the subject is a human.

The language “therapeutically effective amount” of a deprenyl compound,as used herein, refers to an amount of a therapeutic compound sufficientto significantly ameliorate glaucoma or at least one symptom thereof ina subject. “Significant amelioration” includes elimination orsubstantial reduction in severity of one or more symptoms or diagnosticcharacteristics of glaucoma. A “substantial reduction” means at leastabout 5% reduction, more preferably at least about 10% reduction, andmore preferably at. least about 20% reduction in severity of one or moresymptoms or diagnostic characteristics of glaucoma. Thus, atherapeutically effective amount of a therapeutic compound can decreaseintraocular pressure, prevent or delay death of retinal or optic nervecells, improve field of vision or visual acuity, or otherwise ameliorateglaucoma in a subject. One of ordinary skill in the art would be able todetermine such amounts based on such factors as the subject's size, theseverity of the subject's symptoms, and the particular deprenyl compoundor route of administration selected.

I. Deprenyl Compounds

The language “deprenyl compound”, as used herein, includes deprenyl(N,α-dimethyl-N-2-propynylphenethylamine), compounds which arestructurally similar to deprenyl, e.g., structural analogs, orderivatives thereof. Thus, in one embodiment, a deprenyl compound can berepresented by the following formula (Formula I):

in which

R₁ is hydrogen, alkyl, alkenyl, alkynyl, aralkyl, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, or aryloxycarbonyl;

R₂ is hydrogen or alkyl;

R₃ is a single bond, alkylene, or —(CH₂)_(n)-X-(CH₂)_(m);

in which X is O, S, or N-methyl; m is 1 or 2; and n is 0,1, or 2;

R₄ is alkyl, alkenyl, alkynyl, heterocyclyl, aryl or aralkyl; and

R₅ is alkylene, alkenylene, alkynylene and alkoxylene; and

R₆ is C₃—C₆ cycloalkyl or

—C≡CH; or

R₂ and R₄-R₃ are joined to form, together with the methine to which theyare attached, a cyclic or polycyclic group;

and pharmaceutically acceptable salts thereof.

In preferred embodiments, R₁ is a group that can be removed in vivo. Incertain embodiments, R₁ is hydrogen. In other preferred embodiments, R₁is methyl. In certain preferred embodiments, R₂ is hydrogen. In certainpreferred embodiments, R₂ is methyl. In some preferred embodiments, R₃is alkylene, more preferably methylene. In other preferred embodiments,R₃ is —(CH₂)_(n)-X-(CH₂)_(m). In preferred embodiments, R₄ is aryl. Incertain preferred embodiments, R₄ is phenyl. In other preferredembodiments, R₄ is aralkyl. In yet other preferred embodiments, R₄ isalkyl. In still other preferred embodiments, R₅ is alkylene, morepreferably methylene. In certain preferred embodiments, R₆ is

—C≡CH.

In other preferred embodiments, R₆ is cyclopentyl.

In another preferred embodiment, the deprenyl compound has the structure

wherein R₁ is as described above. In other preferred embodiments, thedeprenyl compound is AGN-1133 (N-methyl-N-propynyl-1-indanamine) orAGN-1135 (N-propynyl-1-indanamine). Preferred deprenyl compounds include(−)-deprenyl, (−)-pargyline, (−)-desmethyldeprenyl, and

In another embodiment, a deprenyl compound can be represented by thefollowing formula (Formula II):

in which

R₁ is hydrogen, alkyl, alkenyl, alkynyl, aralkyl, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, or aryloxycarbonyl;

R₂ is hydrogen or alkyl;

R₃ is a bond or methylene; and

R₄ is aryl or aralkyl; or

R₂ and R₄-R₃ are joined to form, together with the methine to which theyare attached, a cyclic or polycyclic group;

and pharmaceutically acceptable salts thereof.

In another embodiment, the deprenyl compound can be represented by thefollowing formula (Formula III):

in which

R₂ is hydrogen or alkyl;

R₃ is a bond or methylene; and

R₄ is aryl or aralkyl; or

R₂ and R₄-R₃ are joined to form, together with the methine to which theyare attached, a cyclic or polycyclic group; and

R₅ is alkylene, alkenylene, alkynylene and alkoxylene;

and pharmaceutically acceptable salts thereof.

In yet another embodiment, the deprenyl compound can be represented bythe following formula (Formula IV):

in which

R₁ is hydrogen, alkyl, alkenyl, alkynyl, aralkyl, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, or aryloxycarbonyl;

A is a substituent independently selected for each occurence from thegroup consisting of halogen, hydroxyl, alkyl, alkoxyl, cyano, nitro,amino, carboxyl, —CF₃, or azido;

n is 0 or an integer from 1 to 5;

and pharmaceutically acceptable salts thereof.

In certain embodiments of the invention, the deprenyl compound is notdeprenyl (including (−)-deprenyl).

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 20 or fewer carbon atoms inits backbone (e.g., C₁-C₂₀ for straight chain, C₃-C₂₀ for branchedchain), and more preferably 10 or fewer. Likewise, preferred cycloalkylshave from 4-10 carbon atoms in their ring structure, and more preferablyhave 5, 6 or 7 carbons in the ring structure. Unless the number ofcarbons is otherwise specified, “lower alkyl” as used herein means analkyl group, as defined above, but having from one to six carbon atomsin its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl”have similar chain lengths. Preferred alkyl groups are lower alkyls. Inpreferred embodiments, a substituent designated herein as alkyl is alower alkyl.

Moreover, the term “alkyl” (or “lower alkyl”) as used throughout thespecification and claims is intended to include both “unsubstitutedalkyls” and “substituted alkyls”, the latter of which refers to alkylmoieties having substituents replacing a hydrogen on one or more carbonsof the hydrocarbon backbone. Such substituents can include, for example,halogen, hydroxyl, carbonyl (such as carboxyl, ketones (includingalkylcarbonyl and arylcarbonyl groups), and esters (includingalkyloxycarbonyl and aryloxycarbonyl groups)), thiocarbonyl, acyloxy,alkoxyl, phosphoryl, phosphonate, phosphinate, amino, acylamino, amido,amidine, imino, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate,sulfonate, sulfamoyl, sulfonamido, heterocyclyl, aralkyl, or an aromaticor heteroaromatic moiety. It will be understood by those skilled in theart that the moieties substituted on the hydrocarbon chain canthemselves be substituted, if appropriate. For instance, thesubstituents of a substituted alkyl may include substituted andunsubstituted forms of aminos, azidos, iminos, amidos, phosphoryls(including phosphonates and phosphinates), sulfonyls (includingsulfates, sulfonamidos, sulfamoyls and sulfonates), and silyl groups, aswell as ethers, alkylthios, carbonyls (including ketones, aldehydes,carboxylates, and esters), —CF₃, —CN and the like. Exemplary substitutedalkyls are described below. Cycloalkyls can be further substituted withalkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substitutedalkyls, —CF₃, —CN, and the like.

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The term “aralkyl”, as used herein, refers to an alkyl or alkylenylgroup substituted with at least one aryl group (e.g., an aromatic orheteroaromatic group). Exemplary aralkyls include benzyl (i.e.,phenylmethyl), 2-naphthylethyl, 2-(2-pyridyl)propyl, 5-dibenzosuberyl,and the like.

The term “alkylcarbonyl”, as used herein, refers to —C(O)-alkyl.Similarly, the term “arylcarbonyl” refers to —C(O)-aryl. The term“alkyloxycarbonyl”, as used herein, refers to the group —C(O)-O-alkyl,and the term “aryloxycarbonyl” refers to —C(O)-O-aryl. The term“acyloxy” refers to —O—C(O)-R₇, in which R₇ is alkyl, alkenyl, alkynyl,aryl, aralkyl or heterocyclyl.

The term “amino”, as used herein, refers to —N(R₈)(R₉), in which R₈ andR₉ are each independently hydrogen, alkyl, alkyenyl, alkynyl, aralkyl,aryl, or R₈ and R₉, together with the nitrogen atom to which they areattached, form a ring having 4-8 atoms. Thus, the term “amino”, as usedherein, includes unsubstituted, monosubstituted (e.g., monoalkylamino ormonoarylamino), and disubstituted (e.g., dialkylamino or alkylarylamino)amino groups. The term “amido” refers to —C(O)-N(R₈)(R₉), in which R₈and R₉ are as defined above. The term “acylamino” refers to—N(R′₈)C(O)-R₇, in which R₇ is as defined above and R′₈ is alkyl.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; and theterm “hydroxyl” means —OH.

The term “aryl” as used herein includes 5-, 6- and 7-membered aromaticgroups that may include from zero to four heteroatoms in the ring, forexample, phenyl, pyrrolyl, furyl, thiophenyl, imidazolyl, oxazole,thiazolyl, triazolyl, pyrazolyl, pyridyl, pyrazinyl, pyridazinyl andpyrimidinyl, and the like. Those aryl groups having heteroatoms in thering structure may also be referred to as “aryl heterocycles” or“heteroaromatics”. The aromatic ring can be substituted at one or morering positions with such substituents as described above, as forexample, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like. Aryl groups can also bepart of a polycyclic group. For example, aryl groups include fusedaromatic moieties such as naphthyl, anthracenyl, quinolyl, indolyl, andthe like.

The terms “heterocyclyl” or “heterocyclic group” refer to 4- to10-membered ring structures, more preferably 4- to 7-membered rings,which ring structures include one to four heteroatoms. Heterocyclylgroups include, for example, pyrrolidine, oxolane, thiolane, imidazole,oxazole, piperidine, piperazine, morpholine, lactones, lactams such asazetidinones and pyrrolidinones, sultams, sultones, and the like. Theheterocyclic ring can be substituted at one or more positions with suchsubstituents as described above, as for example, halogen, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN, or thelike.

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycyclic group can be substituted with such substituents as describedabove, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen,sulfur and phosphorus.

It will be noted that the structure of some of the compounds of thisinvention includes asymmetric carbon atoms. It is to be understoodaccordingly that the isomers arising from such asymmetry are includedwithin the scope of this invention. Such isomers are obtained insubstantially pure form by classical separation techniques and bysterically controlled synthesis

The term “can be removed in vivo”, as used herein, refers to a groupthat can be cleaved in vivo, either enzymatically or non-enzymatically.For example, amides can be cleaved by amidases, and N-methyl amines canbe cleaved by enzymatic oxidation. For example, when deprenyl isadministered to a subject, it is believed, as described infra, that themethyl group can be removed in vivo to yield an active compound. As afurther example, with reference to Formula I, when R₁ is alkylcarbonyl,the resulting amide group can be cleaved in vivo to yield a secondaryamine (e.g., R₁ is converted to hydrogen in vivo). Other groups whichcan be removed in vivo are known (see, e.g., R. B. Silverman (1992) “TheOrganic Chemistry of Drug Design and Drug Action”, Academic Press, SanDiego) and can be employed in compounds useful in the present invention.

II. Pharmaceutical Compositions

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject deprenylcompound from one organ, or portion of the body, to another organ, orportion of the body. Each carrier must be “acceptable” in the sense ofbeing compatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

The stability of deprenyl can be affected by the pH of the medium inwhich the deprenyl is formulated. For example, deprenyl is more stableat a pH in the range of about 3-5 than at a pH of about 7. Therefore,when formulating a deprenyl compound in a pharmaceutical composition, itis preferred that the deprenyl compound be maintained at a suitable pH.In preferred embodiments, a pharmaceutical composition of the inventionhas a pH in the range of about 3 to about 5, more preferably about 3 toabout 4. Furthermore, ethyl alcohol is a preferred solvent for improvingstability of deprenyl. Thus, in certain embodiments, alcoholic oraqueous alcoholic media are preferred for the pharmaceuticalcompositions of the invention.

As set out above, certain embodiments of the present deprenyl compoundsmay contain a basic functional group, such as amino or alkylamino, andare, thus, capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable acids. The term “pharmaceutically-acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ during the final isolation andpurification of the compounds of the invention, or by separatelyreacting a purified compound of the invention in its free base form witha suitable organic or inorganic acid, and isolating the salt thusformed. Representative salts include the hydrobromide, hydrochloride,sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate,palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate,glucoheptonate, lactobionate, and laurylsulfonate salts and the like(see, for example, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm.Sci. 66:1-19).

In other cases, the deprenyl compounds of the present invention maycontain one or more acidic functional groups and, thus, are capable offorming pharmaceutically-acceptable salts withpharmaceutically-acceptable bases. The term “pharmaceutically-acceptablesalts” in these instances refers to the relatively non-toxic, inorganicand organic base addition salts of compounds of the present invention.These salts can likewise be prepared in situ during the final isolationand purification of the compounds, or by separately reacting thepurified compound in its free acid form with a suitable base, such asthe hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptablemetal cation, with ammonia, or with a pharmaceutically-acceptableorganic primary, secondary or tertiary amine. Representative alkali oralkaline earth salts include the lithium, sodium, potassium, calcium,magnesium, and aluminum salts and the like. Representative organicamines useful for the formation of base addition salts includeethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine,piperazine and the like (see, for example, Berge et al., supra).

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the deprenyl compound which produces a therapeuticeffect. Generally, out of one hundred per cent, this amount will rangefrom about 0.01 per cent to about ninety-nine percent of activeingredient, preferably from about 0.1 per cent to about 70 per cent,most preferably from about 1 per cent to about 30 per cent.

Methods of preparing these formulations or compositions include the stepof bringing into association a deprenyl compound of the presentinvention with the carrier and, optionally, one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association a deprenyl compound of the presentinvention with liquid carriers, or finely divided solid carriers, orboth, and then, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A deprenyl compound of thepresent invention may also be administered as a bolus, electuary orpaste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically-acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered deprenylcompound moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the deprenyl compounds ofthe invention include pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredient, the liquid dosage forms may contain inertdiluents commonly used in the art, such as, for example, water or othersolvents, solubilizing agents and emulsifiers, such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (inparticular, cottonseed, groundnut, corn, germ, olive, castor and sesameoils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active deprenyl compound, may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more deprenyl compounds of theinvention with one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the deprenyl compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a deprenylcompound of this invention include powders, sprays, ointments, pastes,creams, lotions, gels, solutions, patches and inhalants. The activecompound may be mixed under sterile conditions with apharmaceutically-acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to adeprenyl compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the deprenyl compound inthe proper medium. Absorption enhancers can also be used to increase theflux of the deprenyl compound across the skin. The rate of such flux canbe controlled by either providing a rate controlling membrane ordispersing the deprenyl compound in a polymer matrix or gel. Devices,including patches, which transdermally deliver a deprenyl compound byiontophoresis or other electrically-assisted methods can also beemployed in the present invention, including, for example, the devicesdescribed in U.S. Pat. Nos. 4,708,716 and 5,372,579.

Ophthalmic formulations, eye ointments, powders, solutions, drops,sprays and the like, are also contemplated as being within the scope ofthis invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more deprenyl compounds of the inventionin combination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject deprenyl compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given alone or as apharmaceutical composition containing, for example, 0.01 to 99.5% (morepreferably, 0.1 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given by formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc.; administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Injection (subcutaneous or intraperitoneal) or topicalophthalmic administration are preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrastemal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular deprenyl compound of thepresent invention employed, or the ester, salt or amide thereof, theroute of administration, the time of administration, the rate ofexcretion of the particular compound being employed, the duration of thetreatment, other drugs, compounds and/or materials used in combinationwith the particular deprenyl compound employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a deprenyl compound of theinvention will be that amount of the compound which is the lowest doseeffective to produce a therapeutic effect. Such an effective dose willgenerally depend upon the factors described above. Generally,intraperitoneal and subcutaneous doses of the compounds of thisinvention for a patient, when used for the indicated anti-glaucomaeffects, will range from about 0.0001 to about 10 mg per kilogram ofbody weight per day, more preferably from about 0.001 mg/kg to about 1mg/kg per day.

If desired, the effective daily dose of a deprenyl compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition).

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition of the invention can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824,or 4,596,556. Examples of well-known implants and modules useful in thepresent invention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4.,486,194, which discloses a therapeuticdevice for administering medicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Thesepatents are incorporated herein by reference. Many other such implants,delivery systems, and modules are well known to those skilled in theart.

It is believed that certain deprenyl compounds can be at least partiallymetabolized in vivo after administration to a subject. For example,(−)-deprenyl can be metabolized by the liver to (−)-desmethyldeprenyl,as well as (−)-methamphetamine and (−)-amphetamine, after oraladministration. The hepatic metabolism of (−)-deprenyl can be inhibitedby administration of a P₄₅₀ inhibitor such as Proadifen. In animal andcell-culture studies, administration of Proadifen reduces the ability of(−)-deprenyl to prevent cell death, but does not block the cell-rescuingactivity of (−)-desmethyldeprenyl. Thus, it is believed that at leastone metabolite of (−)-deprenyl, most likely (−)-desmethyldeprenyl, is anactive compound. It is presently believed that (−)-methamphetamine and(−)-amphetamine are inhibitors of the cell-rescuing activity of deprenylcompounds. It is also believed that monoamine oxidase (MAO, includingboth MAO-A and MAO-B) inhibitory activity is not required for treatmentof glaucoma. Absence of MAO inhibitor activity may in fact provide adrug with fewer side effects. Thus, in certain embodiments, it ispreferred that the deprenyl compound have low MAO inhibitor activity, orbe administered so as to minimize MAO inhibition (e.g., by use of asuitable prodrug or formulation).

In view of the foregoing, it is preferable to administer deprenylcompounds by a route that minimizes metabolism to inhibitor compoundssuch as (−)-methamphetamine and (−)-amphetamine, while allowingmetabolism to active compounds such as (−)-desmethyldeprenyl. Metabolismto an active compound can occur at the desired site of activity, e.g.,in the optic nerve. Thus, prodrugs, which are metabolized to activecompounds, are useful in the methods of the invention.

It has been found that certain deprenyl compounds have greatertherapeutic efficacy (e.g., are effective at lower doses) whenadministered so as to decrease or prevent the “first-pass” effect.Accordingly, intraperitoneal or especially subcutaneous injection arepreferred routes of administration. In preferred embodiments, a deprenylcompound is administered in divided doses. For example, a deprenylcompound can be administered by frequent (e.g., pulsed) injections, orby a controlled infusion, which can be constant or programmably variedas described above. In preferred embodiments in which a deprenylcompound is administered orally, the deprenyl compound can be formulatedto reduce the amount of hepatic metabolism after oral administration andthereby improve the therapeutic efficacy.

In certain embodiments, the deprenyl compounds of the invention can beformulated to ensure proper distribution in vivo. For example, theblood-brain barrier (BBB) excludes many highly hydrophilic compounds. Toensure that the therapeutic compounds of the invention cross the BBB (ifdesired), they can be formulated, for example, in liposomes. For methodsof manufacturing liposomes, see, e.g., U.S. Pat. No. 4,522,811;5,374,548; and 5,399,331. The liposomes may comprise one or moremoieties which are selectively transported into specific cells or organs(“targeting moieties”), thus providing targeted drug delivery (see,e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplarytargeting moieties include folate or biotin (see, e.g., U.S. Pat. No.5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem.Biophys. Res. Commun. 153:1038); antibodies (P. G. Bloeman et al. (1995)FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother.39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J.Physiol. 1233:134); gp120 (Schreier et al. (1994) J. Biol. Chem.269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett.346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273. In apreferred embodiment, the therapeutic compounds of the invention areformulated in liposomes; in a more preferred embodiment, the liposomesinclude a targeting moiety.

The following invention is further illustrated by the following example,which should in no way be construed as being further limiting. Thecontents of all references, pending patent applications and publishedpatent applications, cited throughout this application are herebyincorporated by reference. It should be understood that the animal modelfor glaucoma and optical nerve rescue used in the example is acceptedand that a demonstration of efficacy in these models is predictive ofefficacy in humans.

EXAMPLES

Example

METHODS

All animals were handled according to the Declaration of Helsinki andThe Guiding Principles in the Care and Use of Animals. Underhalothane/nitrous oxide anaesthesia, two groups of adult Strague-Dawleyrats weighing 200-300 gm, eight in each group, were placed in astereotactic instrument and 1.0 micoliter of 3% Fluoro-Gold (FG,Fluorochrome Inc.), a retrograde tracer, was stereotactically injectedbilaterally near to the center of each superior colliculus (Bregmaco-ordinates—5.3 mm, 2 mm lateral and 4.5 mm deep). The injections weredelivered from a 10 microliter Hamilton syringe over a period of fifteenminutes. Slow injections were used to avoid tissue disruption and thelarge volume was employed to insure diffusion would extend over the fulllength of each superior colliculus (SC) (see Tatton et al. ((1991)Neurosc. Letters 131, 179-182) for details and rationale of FG injectionto pre-label the cell bodies of neurons sending axons to a specificBrian structure).

It has been reported that 40-50% of the neurons whose cell bodies arelocated in the retinal ganglion cell layer (RGCL) of the rat send anaxon to the optic nerve and are RGCs (Cowey et al. (1979) Exp. BrainRes. 35, 457-64; Perry V. H. (1981) Neuroscience 6, 931-44: and Lindenet al. (1989) Brain Res. 272, 145-149). The remaining cell bodies in therat RGCL have been shown to be displaced amacrine cells which do notproject to the optic nerve (Perry V. H. (1981) Neuroscience 6, 931-44).Most RGCs (>95%) in rats send their axons to the SC with as many as 10%also sending branches to the lateral geniculate body (Cowey et al.(1979) Exp. Brain Res. 35, 457-64). Hence the FG injections into thesuperior colliculus would be expected to retrogradely-label the cellbodies of RGCs projecting to the superior colliculus (RGC^(SC)s) butwould not label the displaced amacrines in RGCL thereby providing anunambiguous method of RGC^(SC) identification.

Four days were allowed for the retrograde transport of the FG to theRGC^(SC)s cell bodies and then the left optic nerve of the rats wasexposed and crushed by applying the tips of Dumont number 5 forceps tothe nerve immediately behind the globe for ten seconds. Carefulattention was used to avoid the central retinal artery. Each nerve wascrushed three times at the same site. Following the crushes, the nervewas examined with the aid of an operating microscope to ensure that theaxonal component of the nerve was divided into two clearly separatedstumps surrounded by an unbroken dural sheath. Retinal artery patencywas confirmed by direct ophthalmoscopy.

One group of eight rats received (−)-deprenyl (1 mg/kg) byintraperitoneal (IP) injection every two days for a fourteen day periodbeginning at the time of optic nerve crush. The remaining group of eightrats received IP injections of saline on the same schedule. Fourteendays after the optic nerve crush all sixteen rats were euthanized withan overdose of somnotol and perfused transcardially with 4%paraformaldehyde in phosphate buffer. The brains and retinas wereimmersed in 20% sucrose for twenty four hours and then frozen in −70degrees C methylbutane. Serial 10 mm sections were cut through the upperbrain stem and diencephalon of the animals and viewed under fluorescencemicroscopy to determine the location and extent of the stereotacticinjections of FG. Serial 5 mm sections of the retinas were cut and everythird section was Nissl stained. In order to detect even low level FGfluorescence in neuronal cell bodies or processes, the FG labelled brainsections and the retinal sections were examined with the aid of aHamarmatsu intensification camera that allowed the fluorescent images tobe digitized using a Matrox frame grabber controlled by image analysissoftware (Universal Metamorph). This allowed for the creation ofcomputer images of neuronal cell bodies containing FG in the sectionstaken from areas of the stereotactic injections and sections takenthrough each of the retinas.

About 25 sections were chosen randomly from the serial sections takenfrom each retina. Sections were only chosen from the middle 70% of theserial sections where section length exceeded 6 mm. Nissl stained cellbodies in the RGCL were counted at 1000× magnification under oilimmersion on a Leitz Orthoplan microscope. RGCL somata were only countedif they contained a well defined nucleus. The cross sectional length ofthe ganglion cell layer for each section was measured by transferringthe retinal section image into a computer using a CCD camera (Dage Ltd.)and IPPLUS 2.1 software (Media Cybernetics). The number of RGCL neuronalcell bodies (RGCLncbs) per mm length of retinal crossection wascalculated and were pooled for each of the four lesion/treatment groups(uncrushed-saline, uncrushed-deprenyl, crushed-saline andcrushed-deprenyl).

A similar group of sections were randomly chosen from one of the tworemaining series of retinal sections. Those sections were lightly Nisslstained and then were mounted in Fluoromount to allow for counts ofNissl stained RGCLncbs under brightfield microscopy and counts ofRGCLncbs containing FG under fluorescence microscopy from identicalretinal fields. Determination of numbers of RGCLncbs that met the Nisslcriteria for neurons and also contained FG were made by switching backand forth from brightfield to fluorescence microscopy. The ratio of theFG containing cell bodies/Nissl stained neuronal somata in each sectionwas used to determine the proportion of RGCLncbs that were RGC^(SC)s.

Previous studies have shown that the density of RGCs varies from 1600per mm² in the peripheral retina to 2500 per mm² in some parts near thearea centralis (Perry V. H. (1981) Neuroscience 6, 931-44). Since thesections that were counted spanned 70% of the width of each retina, theywould be expected to include different proportions of the central andperipheral retina. This would result in widely varying RGCL ncbs/mm fordifferent Nissl stained sections and marked differences in the numbersof Nissl stained and FG containing cell bodies that would be identifiedas RGC^(SC)s. Accordingly, the distributions of cell body counts weredetermined from the pooled values for each experimental group. Todetermine the statistical significance of any changes in thosedistributions, the count for each section was treated as a single valueand the Kolmogorov-Sminov test (Siegel S. Non-parametric statistics forthe behavioral sciences. In: New York: McGraw Hill Book Company.1956:127-136) was used to compare the values obtained from the fourdifferent experimental groups in a pairwise fashion. TheKolmogorov-Smirnov test is a non-parametric test which does not assumean underlying binomial distribution or that the values are linearlyrelated to each other. The method is optimal for comparing widelydistributed data (see Ju et al. (Exp. Neurol. 126, 233-246) for anexample of its use to determine the significance of changes of pooledvalues taken from large numbers of microscopic sections).

An intensified fluorescent image of a frontal section taken through theSC at 18 days after an injection of FG was examined. Cell bodies andprocesses in all layers of the SC were found to be labelled with FG.Because of the relatively large injection, FG labelling could be seenover the full rostrocaudal length of both SCs and extended to nearbybrainstem and diencephalic structures, including the most caudalportions of the lateral geniculate body. This demonstrated that most, ifnot all, RGC axons projecting to the SC had taken up FG and transportedthe fluorescent marker to their retinal cell bodies. Photomicrographs ofthe same field of a single retinal section viewed alternately underinterference contrast microscopy and fluorescence microscopy showedtypical RGC^(SC)s that were Nissl stained and fluoresced for FG.

FIG. 1 presents box plots of the distributions of the counts of RGCLncbsfor the four experimental groups presented with scales showing thecounts per section or the percent of cell bodies relative to the meanvalue found in the uncrushed saline treated group. There was nostatistical difference between the distributions for uncrushed saline(48.35+/−10.75/mm) and the uncrushed deprenyl (48.39+/−8.48/mm) groups(p>0.05). The distribution for the uncrushed saline group wassignificantly different from the distribution for the crushed salinegroup (22.70+/−7.44/mm, p<0.0001) and the distribution for the crusheddeprenyl group (32.60+/−9.94, p<0.01). Importantly, the distribution forthe crushed deprenyl group was shifted to significantly greater valuesthan that for the crushed saline group (p<0.001). In short, the numbersof neuronal cell bodies/mm in the RGCL of crushed saline retinas werereduced to an average of 46% of those in uncrushed saline retinae and(−)-deprenyl treatment increased that value to an average of 65%. If, aspreviously reported, 40-50% of the neurons in the rat RGCL do not sendaxons to the optic nerve and would not be damaged by the crushes, thenthese values indicate less than 10% survival for RGCs at fourteen daysin the crushed saline retinas and approximately 30-40% survival in thecrushed deprenyl retinas.

To determine whether the joint counts of Nissl and FG cell bodies in theRGCL was a reliable estimate of the proportion of RGCs in the layer, thecounts of Nissl stained cell bodies and FG labelled cell bodies wereplotted as x and y values for varying lengths of retinal sections takenfrom different portions of the differently lesioned and treated retinas(FIG. 2). The Nissl and FG counts were found to covary linearlyindependently of section length or location for the uncrushed saline,uncrushed deprenyl, and crushed deprenyl retinal groups with regressioncoefficients 0.98, 0.96 and 0.90 respectively and y axis intercepts nearzero. The slopes did not differ significantly (p>0.05) for the uncrushedsaline (0.478+/−0.021) and uncrushed deprenyl retinal groups(0.379+/−0.021). The slope of the crushed deprenyl groups(0.219+/−0.019) was significantly different from the uncrushed salineand uncrushed deprenyl groups (p's<0.0001). Since the counts of FGlabelled cell bodies in the crushed saline retinas was very low (averageof 0.66/mm), fitting the data to a linear relationship was not valid.

The mean ratios of FG labelled to Nissl stained somata was 0.425+/−0.045for the uncrushed saline group, 0.388+/0.053 for the uncrushed deprenylgroup, 0.003+/−0.01 for the crushed saline group, and 0.245+/−0.055 forthe crushed deprenyl group. Therefore, by pooling the uncrushed salineand uncrushed deprenyl results, these data estimate that approximately40.7% of the neuronal cell bodies in the RGCL of retinas with uncrushedoptic nerves sent their axons to the SC. These results are consistentwith previous reports that 40-50% of the neurons in the rat RGCL areRGCs which send an axon to the optic nerve and that greater than 95% ofrat RGCs send axons to the SC.

The counts of RGCLncbs and the proportions of FG-labelled cell bodieswere combined to determine the distributions RGC^(SC)s expressed aspercentages of the mean value of the distribution for the uncrushedsaline retinas (FIG. 3). There were no significant differences (p>0.05)in the distributions for the uncrushed saline RGC^(SC) group(100.0+/−22.2%) and the uncrushed (−)-deprenyl RGC^(SC) group(87.0+/−15.2%). In contrast, crushed saline RGC^(SC) group (3.0+/−1.0%,p<0.0001) and the crushed (−)-deprenyl RGC^(SC) group (36.9+/−11.2%,p<0.0001) were distributed differently than the uncrushed salineRGC^(SC)group. The increase in survival in the crushed (−)-deprenylRGC^(SC) group was significantly different from the crushed salineRGC^(SC) group (p<0.001).

EQUIVALENTS

Those skilled in the art will recognize or be able to ascertain, usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A method for treating a subject for elevatedintraocular pressure, comprising: administering a liposome containing atherapeutically effective amount of a deprenyl compound to a subjectsuch that the subject is treated for glaucoma.
 2. The method of claim 1,wherein the deprenyl compound is represented by the structure:

in which R₁ is hydrogen, alkyl, alkenyl, alkynyl, aralkyl,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, or aryloxycarbonyl; R₂ ishydrogen or alkyl; R₃ is a single bond, alkylene, or—(CH₂)_(n)—X—(CH₂)_(m); in which X is O, S, or N-methyl; m is 1 or 2;and n is 0,1, or 2; R₄ is alkyl, alkenyl, alkynyl, heterocyclyl, aryl oraralkyl; and R₅ is alkylene, alkenylene, alkynylene and alkoxylene; andR₆ is C₃-C₆ cycloalkyl or —C≡CH; or R₂ and R₄-R₃ are joined to form,together with the methine to which they are attached, a cyclic orpolycyclic group; and pharmaceutically acceptable salts thereof.
 3. Themethod of claim 2, wherein R₁ is a group that can be removed in vivo. 4.The method of claim 2, wherein R₁ is hydrogen.
 5. The method of claim 2,wherein R₁ is alkyl.
 6. The method of claim 5, wherein R₁ is methyl. 7.The method of claim 2, wherein R₂ is methyl.
 8. The method of claim 2,wherein R₃ is methylene.
 9. The method of claim 2, wherein R₄ is aryl.10. The method of claim 2, wherein R₄ is phenyl.
 11. The method of claim2, wherein R₅ is methylene.
 12. The method of claim 2, wherein R₆ is—C≡CH.
 13. The method of claim 2, wherein the deprenyl compound has thestructure

wherein R₁ is hydrogen, alkyl, alkenyl, alkynyl, aralkyl, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, or aryloxycarbonyl.
 14. The method ofclaim 2, wherein the deprenyl compound is represented by the structure:

in which R₁ is hydrogen, alkyl, alkenyl, alkynyl, aralkyl,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, or aryloxycarbonyl; R₂ ishydrogen or alkyl; R₃ is a bond or methylene; and R₄ is aryl or aralkyl;or R₂ and R₄-R₃ are joined to form, together with the methine to whichthey are attached, a cyclic or polycyclic group; and pharmaceuticallyacceptable salts thereof.
 15. The method of claim 2, wherein thedeprenyl compound is represented by the structure:

in which R₂ is hydrogen or alkyl; R₃ is a bond or methylene; and R₄ isaryl or aralkyl; or R₂ and R₄-R₃ are joined to form, together with themethine to which they are attached, a cyclic or polycyclic group; and R₅is alkylene, alkenylene, alkynylene and alkoxylene; and pharmaceuticallyacceptable salts thereof.
 16. The method of claim 2, wherein thedeprenyl compound is represented by the structure:

in which R₁ is hydrogen, alkyl, alkenyl, alkynyl, aralkyl,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, or aryloxycarbonyl; A is asubstituent independently selected for each occurence from the groupconsisting of halogen, hydroxyl, alkyl, alkoxyl, cyano, nitro, amino,carboxyl, —CF₃, or azido; n is 0 or an integer from 1 to 5; andpharmaceutically acceptable salts thereof.
 17. The method of claim 1,wherein the deprenyl compound is (−)-deprenyl.
 18. The method of claim1, wherein the deprenyl compound is (−)-pargyline.
 19. The method ofclaim 1, wherein the deprenyl compound is (−)-desmethyldeprenyl.